Activation of Icrac in wild-type DT40 cells by different agents. Voltage-clamped membrane currents measured at room temperature in normal Ringer's solution. Cells were held at 0 mV, and currents were activated by 160-ms duration voltage steps between –100 and 90 mV in 10-mV steps. I-V curves were measured in the steady state of activated Icrac currents between 2 and 10 min after cell stimulation. Currents activated at −100, −30, 0, 30, and 90 mV are shown. (A) Control cell treated with delivery vehicle alone (0.1% DMSO). (B) Treated with 1 μM extracellular TG. (C) Treated with 0.5 μM extracellular ionomycin. (D) Treated with 60 μM IP₃ delivered by diffusion from the lumen of the tight-seal electrode. (E) Treated with a 1:400 dilution of extracellular M4, a specific BCR antibody. To the right of each current set is that cell's I-V curve measured after chemical activation. The current is the mean amplitude measured in the 60–160-ms interval after voltage step onset. To compare among cells of varying size, the current amplitude in each cell was normalized by the cell's membrane surface, which was measured as membrane capacitance. Every one of the drugs tested activated the Icrac current, which is defined by its characteristic inward rectifying features.

Ion selectivity of Icrac in DT40 cells treated with 60 μM intracellular IP₃. (A) Steady-state current amplitudes measured in the same cell between –100 and 90 mV in the sequential presence of extracellular Ringer's solution containing 150 mM Na⁺ and either free of divalent cations (squares; with 2 mM EGTA), with 10 mM Ca²⁺ (triangles), or with 10 mM Ba²⁺ (circles). (B) I-V curve of Icrac in different cells measured in the presence of 150 mM Na⁺ and either 10 mM Ca²⁺ (triangles) or 10 mM Ca²⁺ with 2 mM of added Cd²⁺ (diamonds).

Activation of Icrac in DT40 cells by 60 μM intracellular IP₃. Voltage-clamped membrane currents measured at room temperature in cells bathed with normal Ringer's solution. Cells were held at 0 mV. Currents activated at −100, −30, and 0 mV are shown. To the right of each current set is that cell's steady-state I-V curve after activation. (A–D) Blnk- (A), Lyn- (B), Btk- (C), and Syk-deficient (D) cells. (E) Cell deficient in both Lyn and Syk. The characteristic inwardly rectifying Icrac current is completely absent only in cells lacking both Lyn and Syk tyrosine kinases.

Dependence of Icrac amplitude on intracellular IP₃ concentration in wild-type and mutant cells. Current amplitude measured at −100 mV after cell activation. Closed circles are the mean (±SEM [error bars]) amplitude measured in wild-type (wt) cells, and open circles are data measured in Lyn- and Syk-deficient cells. Lyn- and Syk-deficient cells were not activated even at the highest IP₃ concentration tested.

Activation of Icrac in DT40 cells with 1 μM extracellular TG. Voltage-clamped membrane currents measured in cells bathed in normal Ringer's solution. Currents activated at −100, −30, 0, 30, and 90 mV are shown. To the right of each current set is the steady-state I-V curve for that cell. (A) Wild-type (wt) cell. (B) Lyn- and Syk-deficient cell. In the absence of tyrosine kinases, Icrac current is not activated by TG.

Effects of 1 μM TG on cytoplasmic free Ca²⁺ in populations of normal and mutant DT40 cells. Cytoplasmic Ca²⁺ was measured with the fluorescent Ca²⁺ indicator Indo1. An increase in the 400:500-nm emission intensity ratio reflects a rise in cytoplasmic Ca²⁺. Cells were suspended either in normal Ringer's solution (with 1 mM Ca²⁺) or in Ca²⁺-free Ringer's solution (with 4 mM EGTA). (A) In wild-type (wt) cells, TG application in normal Ringer's solution caused a sustained elevation of cytoplasmic Ca²⁺. (B) In the presence of EGTA, the TG-dependent Ca²⁺ rise was transient, and, after reaching a peak, it returned to nearly the starting value despite the continuous presence of TG. (C and D) In Lyn- and Syk-deficient cells, TG caused a nearly identical response in the presence of Ca²⁺ (C) or in its absence (D). The response was a small, transient rise in free Ca²⁺ that arises from the TG-dependent release of Ca²⁺ from intracellular stores.

Restoration of TG-dependent Icrac activation by Lyn and/or Syk homologous expression in lyn/syk^−/− cells. Cytoplasmic Fura2 emission fluorescence intensity ratio at 340 and 380 nm was measured in individual cells and averaged. (A) Wild-type cells stimulated by 1 μM TG in a Ca²⁺-free Ringer's solution (with 4 mM of added EGTA) demonstrated a transient release of Ca²⁺ from the ER stores. Switching to an extracellular solution containing Ba²⁺ added to the EGTA-containing Ringer's solution (10 mM Ba²⁺ added, yielding 8 mM of free Ca²⁺) caused a change in Fura2 fluorescence as a result of Ba²⁺ influx through activated Icrac channels (mean of 42 cells). (B) Lyn- and Syk-deficient cells. TG caused the release of intracellular Ca²⁺ stores, but the addition of extracellular Ba²⁺ did not cause changes in intracellular Fura2 fluorescence because Ba²⁺ influx does not occur in the absence of active Icrac channels (mean of 19 cells). Panels on the right illustrate results from measurements in Lyn- and Syk-deficient cells reconstituted to express Lyn or Syk alone or together. Because the heterologous protein was in fusion with GFP, cells in a field of view were identified as kinase expressing and GFP positive (closed circles) or as lyn/syk^−/− and GFP negative (open circles). The response of Lyn- and Syk-deficient cells is the same in all panels (C, mean of 35 cells; D, 20 cells; E, 25 cells). TG causes intracellular Ca²⁺ release, but Ba²⁺ cannot enter through closed Icrac channels. On the other hand, the successful expression of tyrosine kinases (C, Lyn alone, mean of nine cells; D, Syk alone, three cells; E, both, three cells) allows Icrac activation by released Ca²⁺, and, thus, changes in Fura2 fluorescence are observed in these cells when the bathing solution is switched to one containing Ba²⁺. In E, the dotted line identifies the moment Ba²⁺ was first added to the bathing solution. It corresponds to the transition from gray to black in the horizontal bar.

The specific tyrosine kinase inhibitor LavendustinA blocks the activation of Icrac in wild-type DT40 cells. (A and B) Electrophysiological assay. Membrane voltage was held at 0 mV, and the difference in current amplitude in response to −90-mV steps was measured repeatedly every 3 s and box car averaged over three adjacent data points. In the graph, time 0 is the moment whole cell mode was first achieved. 90 s thereafter, the cell bathing solution was switched to one containing 1 μM TG. (A) In a normal cell, TG slowly activated an inward current that reached a final, stationary value ∼60 s after TG superfusion began (inset). The I-V curve of the TG-dependent current was measured as the difference in I-V curves acquired at time 0 s and at 85 s after initiating TG superfusion. This I-V curve is typical of Icrac. (B) TG, in contrast, did not cause any current change in cells treated with LavendustinA (inset). Because there are no TG-dependent currents in LavendustinA-treated cells, the difference in I-V curves acquired at time 0 and at 85 s after initiating TG superfusion is essentially flat. (C and D) Ca²⁺ imaging assay. (C) In Lyn- and Syk-deficient cells, stimulation with 1 μM TG in a Ca²⁺-free Ringer's solution (with 4 mM EGTA) caused a rise in intracellular Ca²⁺ (increase in Fura2 fluorescence F340/F380 emission intensity), but adding Ba²⁺ tothe EGTA-containing Ringer's solution (10 mM Ba²⁺ added, yielding 8 mM of free Ca²⁺) to the extracellular medium did not cause further changes in Fura2 fluorescence (mean of 45 individual cells). (D) Wild-type cells were incubated for 10 min with 10 μM LavendustinA and assayed in the continuing presence of the drug. These drug-treated cells exhibit the same functional behavior as Lyn- and Syk-deficient ones: Icrac is not activated, and Ba²⁺ added after TG stimulation does not cause the enhancement in Fura2 emission intensity observed in wild-type cells. Error bars represent SEM.

The specific Src tyrosine kinase inhibitor PP2 blocks the activation of Icrac in wild-type DT40 cells. Cytoplasmic free Ca²⁺ measured in a suspension of cells loaded with Indo1. (A) Normal cells suspended in Ca²⁺-free Ringer's solution (4 mM EGTA) respond to stimulation by 1 μM TG with a small, transient increase in free Ca²⁺. Addition of external Ca²⁺ to the EGTA-containing Ringer's solution (5 mM Ca²⁺ added, yielding 1 mM of free Ca²⁺) causes a large increase in cytoplasmic Ca²⁺ as a result of cation influx through activated Icrac channels. (B) Lyn- and Syk-deficient cells. In response to 1 μM TG in a Ca²⁺-free Ringer's solution, there is a small, transient increase in Ca²⁺ similar to that in wild-type (wt) cells. The addition of extracellular Ca²⁺ causes a small additional increase in free Ca²⁺ that is smaller in extent than that observed in wild-type cells. (C) Wild-type cells treated with 20 μM PP2. The functional behavior of these cells is indistinguishable from the Lyn- and Syk-deficient ones. In the absence of extracellular Ca²⁺, TG causes a small and transient Ca²⁺ release from intracellular stores. Icrac is not activated, and, therefore, the addition of extracellular Ca²⁺ does not cause the large and sustained increase in free Ca²⁺ observed in untreated normal cells.

Biochemical analysis of Lyn and Syk autophosphorylation activity in wild-type and mutant cells. Analysis of the expression of STIM1 and Orai1 in wild-type and mutant cells. (A) BCR- and TG-stimulated Lyn/Syk activity in DT40 cells. Wild-type (wt) or Lyn- and Syk-deficient cells were stimulated with anti-BCR antibody (M4) or with TG. Western blots of immunoprecipitates of tyrosine phosphoproteins isolated from the cells before and 60 or 300 s after TG or the M4 stimulation (as labeled). The same blot was successively probed with antibodies specific for the activation loop in Syk (P-Y352) or Src (P-Y416) kinases. The extent of antibody binding was quantified in relative units of activity (phosphorylation index). The levels of IgG are also shown to demonstrate that the total amount of tyrosine-phosphorylated protein in each lane is similar. The bar graphs are means ± SEM (error bars) of the phosphorylation index in five different experiments measured from the absolute values of gray level (at 10-bit resolution) in the original blots. The images displayed for each ofthe various antibodies tested were independently adjusted with respect to brightness and contrast to bring forth the individual bands (Photoshop [Adobe] at 8-bit gray level resolution). Therefore, visual perception of the band intensity in the images is skewed and does not necessarily match the analytical bar graph. (B) Chemiluminescent image of a Western blot of wild-type and mutant cells assayed for the protein level of STIM1 and tubulin. SDS-PAGE–separated total protein samples were blotted and subsequently identified with specific primary antibodies. The level of expression was determined by the gray level of chemiluminescent images of the identified protein bands. Within the resolution of the technique, STIM1 protein levels are not different in wild-type and mutant cells. (C) Images of ethidium bromide–stained DNA bands in an agarose gel. DNA was obtained by PCR amplification of cDNA using primers specific for STIM1, Orai11, and β-actin. PCR reactions were performed in serially diluted (1:5) samples of the same starting cDNA. After identical PCR reactions, DNA products were size separated by gel electrophoresis, and the gel was stained with ethidium bromide. DNA levels were assessed by the gray level in the images. Within the resolution of the technique, STIM1 and Orai11 mRNA levels are not different in wild-type and mutant cells.